Abstract The goal of the proposed study is to demonstrate the viability of a novel Piwi-interacting RNA (piRNA)-based therapeutic strategy for the treatment of hepatocellular carcinoma (HCC). piRNAs are an abundant class of small (26-31 nt) non-coding RNAs with approximately 23,000 known human members and play a pivotal role in the maintenance of genomic stability in germ cells via epigenetic silencing of transposable elements (TE). Recent evidence from our group and others has suggested that piRNAs can also induce DNA methylation at non-TE protein-coding genes, including cancer-related genes in somatic cells. However, the role of piRNAs in human cancers has not been fully studied and no previous publication has explored the use of piRNA as a therapeutic tool. Our previous analyses have detected marked differences in expression of several piRNAs between normal liver tissue and HCC tissue as well as anti-cancer effects of an exogenously modulating expression of one of these identified piRNAs. In the proposed study, we seek to expand upon these important findings by further examining the potential anticancer impact in vivo for the candidate piR-37213. Specifically, we will first examine the anti-tumor effect of piR-37213 on HCC cells in vivo using an orthotopic mouse xenograft model (Specific Aim 1), which allows us to test whether the observed anti-proliferative effects in vitro can be replicated in live animals. We will also examine molecular mechanisms accounting for the anti-tumor effect of piR-37213 (Specific Aim 2). In this Aim, we will first perform a genome-wide expression profiling study to reveal the gene regulation network and global impact of piR-37213, and confirm these findings with relevant in vitro assays if applicable. Next, we will perform a RNA pull-down assay to explore potential piRNA binding sequences. This study represents the key first step in the development of an innovative, piRNA-based therapeutic strategy that may be utilized as a personalized treatment approach via the restoration of normal piRNA expression on an individual basis. Knowledge obtained from this study will advance our understanding of a novel mechanism underlying HCC biology, expand knowledge of the role and function of piRNA in tumorigenesis, and provide new avenues of research that can be exploited to facilitate the development of novel therapeutic strategies.